1. Detectors for light microscopy Marcin Barszczewski PhD 1

2. Single Molecule Detection Low Light Imaging Live cell confocal Application examples TIRF Ion signalling Selective Plane Illumination Microscopy (SPIM) Intracellular luminescence Cell Motility FRET Single photon counting Fluorescence Correlation Spectroscopy (FCS) Super-resolution PALM/ STORM Adaptive optics 3. How to detect light from your samples? - PMTsPoint detectors - APDs1D, Low QE, uniformity, repeatability,Speed, low noise factor (PMTs)high noise factor (APD) - CCDs Array (2D) detectors - CMOSHigh sensitivity, low noise, 2D Historically low speed, dynamic range, packaging 4. Scientific imaging trade-offsTodays imaging detectors exhibit trade-offs between key performance parameters Low noise/sensitivity Speed Wide dynamic range High resolution Large field of view 5. When do we need sensitivity? Low dye concentrations / single molecule Short exposures / fast frame rates High photon loss / rejection Lower excitation power Greater magnifications Low quantum yield / Raman scatter 6. What makes a detectorsensitive?Two key parameters Quantum Efficiency Noise floorDetectors must be designed to ensurethese parameters are optimized. 7. Typical Quantum Efficiency CurvesBack-illuminated 100Virtual Phase 90MicroLens Front-illuminated 80 front-illuminated 70Front-illuminatedQE (%) 60ICCD 50 40 30 20 100 200 300 400500 600700 8009001000Wavelength (nm) 8. Making sense of sensitivityShot Noise Read noise VariationUsual camera detection limit. Dark noiseDependent on temperatureAverage Shot Noise Signal Intensity QE and signal dependent. Noise Floor (Read Noise and Dark Noise) 9. Camera-based imaging technologies Sony interline EMCCD sCMOS Sensor format1.4 MP1 MP (max.)5.5 MP Pixel size 6.45 m8 to 24 m 6.5 m Max. frame rate12 fps @ 20MHz > 30 fps 100 fps Read noise4 8 e- Negligible (90% (BI)~ 57% (FI) (excellent red response) Dynamic range ~ 3,000:1 ~ 8,500:130,000:1 (@ 11 frames/sec) (@ 30 frames/sec)(@ 30 frames/sec) Darkcurrent (TE cooled)0.0003 e/pix/sec0.001 e/pix/sec0.07 e-/pix/sec@ -55 0C@ -85 0C @ -30 0C 10. Frame transfer EMCCD gain register 11. Effect of EM Gain on signal-to-noise EMCCD Gain Gain x1 Gain x10 Gain x100 Gain x500 12. CMOS vs. CCD Architecture 13. What makes a detectorsensitive?Two key parameters Quantum Efficiency Noise floor 14. Primary sources of noise within imaging sensors1. READ NOISE- Caused by electronic noise in the CCD output transistor and in theexternal circuitry2. DARK CURRENT- Caused by thermally generated electrons in the CCD3. PHOTON NOISE / SHOT NOISE- It is due to the fact that the CCD detects photons+ other noise types 15. CCDs - reduced read noise1MHz (offering ~ 1 fps) - 2.4 e- read noisebut slower frame rate - Read Noise is a fundamental trait of CCDs - Read noise can be accounted and corrected - Its influence on images 20MHz (offering ~ 11 fps) 5.5 e- read noise can be decreased by reducing frame rates 16. Impact of extensive cooling on weak signals-70 0C -95 0C - All CCDs build up darkcurrent whether the CCD isbeing exposed to light ornot- The rate of dark currentbuild up can be reduced bya factor of 100 or more bycooling the CCD- The remaining dark current Extremely weak signal low-light is subtracted from an image luminescence experimentusing dark frames. High EM Gain 17. External noise source - background photon Sources? Out of focus fluorescence background counter withconfocal, TIRF, SPIM Non-optimal optical filters Stray background light Non-specific binding of fluorophore. 18. ApplicationConsiderations- which camera for what application? 19. Scientific imaging trade-offsTodays imaging detectors exhibit trade-offs between key performance parameters Low noise/sensitivity Rapid frame rates Wide dynamic range High resolution Large field of view 20. Scientific CMOS (sCMOS)is unique in simultaneously offering: Extremely low noise (without multiplication) Rapid frame rates Wide dynamic range High QE High resolution Large field of view 21. TheElectron MultiplyingCharge Coupled Device(EMCCD) Eliminates read noise detection limit High Quantum Efficiency Fast frame rates with the lowest noise 22. Single Molecule Detection extremely low light regime - a back-illuminated EMCCDdomain pushing typical exposure times even shorterLive cell imaging sCMOS greater flexibility with FOV, resolution, speed Some low light modalities will still need the sensitivity ofEMCCD, e.g. spinning disk confocal 23. EMCCD vs. sCMOS image comparison490 68 8photonsphotons photonsper pixelper pixel per pixelsCMOS 2x2 binned(13 m)EMCCD(13 m) 24. Field of View comparisons sCMOS Sony ICX285 interline Field of view comparison of two technologies; x60 magnification; 1.25 NA; 5.5 megapixelsCMOS vs 1.4 megapixel interline CCD (each have ~ 6.5 m pixel pitch). 25. SPIM (Selective Plane Illumination Microscopy) Neo sCMOS Dr. Lars Hufnagel, Developmenta l Biology Unit, EMBL Heidelberg. Resolution Field of View Speed Optical sectioning even with lenses that have a large workingdistance and a relatively low numerical aperture Especially well suited for the investigation of large samples (e.g.embryos) to study features such asgrowth, migration, morphological changes and gene expressionpatterns, that require high resolution, while being extended over alarge volume. Single plane illumination significantly reduces Mouse Embryophotobleaching/phototoxicity 26. Optical cross-sections through a developing Drosophila melanogaster embryo in stage 5/6. TwoNeos are used to capture this 3-D structure and one of these can be captured every 20seconds. 26 27. Thank you foryour attention!27